1. Field of the Invention
The present invention relates to a method and a recombinant vector for coexpressing IL-12 (interleukin-12) and IL-23 (interleukin-23), and a pharmaceutical anti-tumor composition.
2. Background of Technique
Cancer is a disease in which human normal cells escape a cell-cycle control due to genetic mutations caused by various carcinogens and exhibit immortal proliferation, resulting in infiltration into surrounding tissue and metastasis into other internal organs or tissues through blood and lymphatic vessels. Because most cancers were diagnosed after the occurrence of metastasis into other organs or tissues without apparent symptoms, surgical operations, radiotherapy and anti-tumor chemotherapy in use as current therapies have exhibited many limitations. Therefore, novel approaches for effective therapy different from current therapies have been now needed urgently. In this connection, gene therapy and immunotherapy have been actively researched as novel therapeutic methods for various types of cancer diseases1-3.
Immunotherapy of cancer is aimed to specifically remove cancer cells by boosting the innate immune system. In the early 20th century, Paul and his colleagues4 asserted that human innate immune system could recognize and remove tumors, suggesting the possibility of anti-tumor immunotherapy for the first time and in the middle 20th century, Gross and his colleagues5 first demonstrated Paul's hypothesis using animal models.
Thereafter, the theory underlying immune surveillance was founded by Burnet and Thomas6-8. Due to the rapid development of genetic engineering and molecular immunology, immune cells and their action mechanisms inducing anti-tumor immune response were revealed, resulting in new turning point in the field of anti-tumor immunotherapy. Much interests and researches have been progressed up to date.
In spite of the rapid development and many efforts, immunotherapy has revealed many limitations to treat cancers. Because cancer cells could avoid ingeniously the immune system through various strategies and escape from activated anti-tumor immune response, they could proliferate continuously in the event. To be free from these limitations, the immune response against tumors has been induced by introducing immunostimulatory cytokine genes directly into cancer cells for producing and secreting cytokines in cancer cells per se, such that cancer cells are specifically removed.
The genes encoding immunostimulatory molecules with anti-tumoric effects include IL-29-10 (interleukin-2), IL-411-12, IL-713, IL-1214, G-CSF (granulocyte colony-stimulating factor)15, GM-CSF (granulocyte macrophage colony-stimulating factor)16 and IFN-γ17 (interferon-γ). Of them, IL-12 is a heterodimeric protein consisting of p35 and p40 and is secreted from antigen-presenting cells (APC) such as monocytes, macrophages, and dendritic cell. In addition, it activates cancer-killing cytotoxic T-lymphocyte (CTL) and natural killer (NK) cells to induce secretion of IFN-γ and elevation of their oncolytic activity. It has been also known that IL-12 stimulated naive CD4+ lymphocytes to differentiate into T-helper 1 (Th1) cells, resulting in induction and enhancement of cell-mediated immune responses responsible for anti-tumor activities, and inhibited cancer metastasis. On the basis of the findings described above, our laboratory has reported anti-tumor effects of IL-12 using YKL-1 (Ad-ΔE1B55) as the oncolytic adenovirus with the deleted E1B 55 kDa gene.
IL-23 having a similar structure with IL-12 is composed of p40 and p19 as a member of IL-12 superfamily, and is secreted from antigen-presenting cells (APC) such as monocytes18, macrophages19 and dendritic cell20. It has been known that IL-23 activated APC and induced not only the secretion of IFN-γ and IL-1220 but also the proliferation of memory T-cells and the secretion of IFN-γ20. Especially, IFN-γ induced by IL-12 and IL-23 promotes the expression of major histocompatibility complex (MHC) in APC and increases the antigen-presenting ability. In this regard, IL-23 not only induces the activation of CTL and helper T-lymphocytes through giving immunogenicity to cancer cell, but also increases the oncolytic activity of NK cells. Given the reports up to date, it is expected that anti-tumor effects would be enhanced where both IL-12 and IL-23 having similarities with each other in terms of structure and function act simultaneously. That is, the activation action of a pair of IL-12 and IL-23 on APC becomes more prominent than alone action of either IL-12 or IL-23, and in turn the pairing action induce the secretion of not only IL-12 and IL-23 but also IFN-γ more strongly, contributing to greater influence on all molecular events underlying anti-tumor effect relating to IL-12 and IL-2321.
For applying effectively adenoviral gene therapy for cancers to practical therapeutics, the development of adenovirus having both the specificity to kill selectively cancer cells without side effects on surrounding normal cells and the capability to kill effectively cancer cells is indispensable. However, because 1st replication-incompetent adenoviruses in which the E1A gene essential for adenovirus replication was deleted, exhibited the infectiveness in only the first generation, they induced anti-tumor activity solely in infected cells and a very small number of surrounding cells, rendering them to have serious problems in clinical applications. To overcome such problems, our laboratory reported both cancer-specific proliferation and oncolytic effects through the development of the E1B 55 kDa-deleted tumor-specific adenovirus, YKL-1 (Ad-ΔE1B55), which could be proliferated selectively in cancer cells lacking functional p53, tumor-inhibitory protein24. The oncolytic adenovirus which could proliferate selectively in the cancer cells and kill them, exhibited therapeutic effects in primarily infected cells and in turn proliferated adenoviruses infected and killed surrounding tumor cells. These features lead to dramatic cancer treatment efficacy with little or no adverse effects due to their incompetence of replication in normal cell. However, the proliferation of YKL-1 adenovirus was restricted relatively as compared with wild type (WT) adenovirus, and its cytotoxicity was reduced significantly, resulting in low anti-tumor effects25.
To augment such lower cell-killing ability of YKL-1 adenovirus, our laboratory had developed and reported in vivo and in vitro excellent anti-tumor effects of Ad-ΔB7 adenovirus showing enhanced cell-killing potential through the deletion of E1B 19 kDa region and significantly improved oncolytic activity through substituting Gly (G) for Glu (E) in 45 position within CR1 region and seven GGGGGGG for seven DLTCHEA in CR2 region. That is, the Ad-ΔB7 adenovirus has not only enhanced oncolytic activity against cancer cells due to increased apoptosis-inducing potential by the deletion of the E1B 19 kDa region to inhibit apoptosis, but also improved oncolytic selectivity by the deletion of the Rb-binding region in the E1A gene and substitution with Gly residue to enable it to replicate only in Rb-mutated cancer cells.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.